Factor IX Bm Kiryu: a Val-313-to-Asp substitution in the catalytic domain results in loss of function due to a conformational change of the surface loop: evidence obtained by chimaeric modelling.

Abstract

Factor IX Kiryu is a naturally occurring mutant of factor IX that has 2.5% coagulant activity, even though normal plasma levels of factor IX antigen are detected. Factor IX Kiryu was purified from a patient's plasma by immunoaffinity chromatography with a calcium-dependent anti-factor IX monoclonal antibody column. It was cleaved normally by factor XIa in the presence of Ca2+, yielding a two-chain factor IXa. However, the resulting factor IXa showed only 1.5% of the normal factor IXa in terms of factor X activation in the presence of factor VIII, phospholipids, and Ca2+, and had 20% of the normal esterase activity for Z-Arg-p-nitrobenzyl ester. Therefore factor IXa Kiryu showed the defect of the catalytic triad or primary substrate binding site as well as defective interaction with factors VIII/X. Single-strand conformational polymorphism analysis and DNA sequencing of the amplified DNA revealed a missense point mutation, a T-to-A substitution at nucleotide number 31,059 of the factor IX Kiryu gene. This mutation resulted in the amino acid substitution of Val-313 by Asp in the catalytic domain. Restriction enzyme analysis of the amplified DNA showed that the mutation was inherited from the patient's mother. The chimaeric method was employed to construct a model of the serine protease domain of factor IXa, and the resultant model suggested that the Val-313 to Asp substitution altered the conformation of the substrate-binding site. These data combined with our previous findings on a Gly-311-to-Glu mutant of factor IX suggest that the loop conformation from Gly-311 to ARg-318 is important for the expression of coagulant activity.